Electrical stimulation of the heart is commonplace in the treatment of cardiac arrhythmias. Implantable cardioverter/defibrillators is now a $1 billion industry in the U.S., with approximately 170,000 shocks delivered in people per year. Yet the fundamental mechanisms by which electrical fields interact with myocardial cells are still largely unknown. In the first (previous) three years of this project, the applicant has experimentally verified new theories for the electrical excitation of cardiac muscle in the vicinity of the stimulating electrodes, using optical measurements of cellular transmembrane potentials, and is working to test a theoretical model of field excitation of single cardiac cells. Nevertheless, the detailed biophysical mechanisms of interaction between electric fields and tissues in regions far from the electrodes are still largely unknown. Theories regarding possible facets of the excitatory process are quite advanced, and suggest that the tissue architecture may be the key player in determining the tissue response. However, at the present time experimental validation is still lacking. The central theme of this continuing five year application is the experimental investigation of the role of tissue architecture in the electrical excitation of cardiac muscle by applied electrical fields. The focus of attention in this project will shift away from amphibian heart, as studied in previous years, towards mamalian heart. The applicant proposes to utilize microlithographic technology to design patterned substrates of any shape on which cultured neonatal rat heart cells will be grown as cell strands. Using this approach, the applicant can then investigate tissue architectures of any desired topology. Features to be investigated include resistive discontinuities between cells, nonuniformities in electric field, strand boundaries, strand angle, strand curvature, strand branching, and changes in strand cross-sectional area. Voltage-sensitive dyes will be used to record the spatial patterns of transmembrane potential response to electric fields under each of these various conditions. Computer modeling will allow quantitative comparisons between experiment and current-day theoretical concepts. In this manner, the applicant will attempt to develop a comprehensive theoretical description of the influence to tissue architecture on the cellular membrane response to stimulating electric fields, component by component, so that the response of realistic tissue architectures can be better analyzed, understood, and ultimately, predicted.